Figure 1.

Hypoxia upregulates Vav1 through inhibition of lysosome-mediated protein degradation. (A) Human umbilical vein endothelial cells (HUVEC)s were cultured either in 20% or 1% O₂ for 5 h, followed by Western blot analysis for the indicated proteins. (B) qPCR analysis of Glut1, VEGF and Vav1 in HUVECs cultured in 1% O₂ for 0, 1, 2, or 3 h. * p < 0.05 compared to corresponding time 0 in each group (mean ± SD). (C) The levels of Vav1 and HIF-1α were measured by Western blot of HUVECs transduced with HIF-1α or scramble shRNA expressing Lentivirus for 24 h. (D) HUVECs were incubated in normoxic and hypoxic environments for 5 h in the presence or absence of 10 μM cycloheximide. Protein levels were measured by Western blot from the total lysate. (E) HUVECs were incubated in 20% O₂ for 5 h, incubated in 20% O₂ for an hour and then moved to 1% O₂ for 4 h, incubated in 1% O₂ for 5 h. The level of Vav1 in the lysate was compared to HUVECs incubated in 20% O₂ in the presence of chloroquine (CQ) at 50 μM for 5 h in order to identify whether Vav1 is affected by lysosomal inhibition. (F) HUVEC were incubated in normoxic or hypoxic conditions for 4 h in the presence of either vehicle control, 5 μM of MG-132, or 50 μM of chloroquine (CQ). The total lysate was subjected to Western blotting to measure Vav1 protein levels. (G) Western blot analysis of Vav1 levels in HUVECs cultured in either 20% or 1% O₂ in the absence or presence of Bafilomycin A (Baf A) at 100 nM for 5 h. (H) Immunofluorescent staining for Vav1 (red) and Cathepsin D (green) in HUVECs were imaged by an LSM780 confocal microscope. Each experiment was repeated at least ten times and representative images are shown. Mean ± SD, * p < 0.05, ** p < 0.01. The whole western blot images please find in Figure S1.